It is increasingly apparent that the low sort speed, lack of usable parameters and low sensitivity of the cell sorter at the Trudeau Institute is preventing the investigators at the Institute from completing the goals of their NIH funded projects. With the ongoing expansion of the Institute and the recruitment of new faculty, it has become clear that this instrument cannot be made to meet the swelling demand for complex, multi-laser cell sorting and analysis. Therefore, to meet the requirements of the Institute's current and newly-recruited faculty, this proposal requests funds to purchase a Cytomation MoFlo cell sorter. This new sorter will be the central component of a newly constructed centralized FACS facility and will be essential to achieving the goals of 24 currently active NIH grants, as well as 15 proposals under review. Many of these programs are at the stage where the next critical experiments require the purification of large numbers of relatively rare cells with complex phenotypes such as MHC Class I and Class II tetramer-staining T cells, antigen specific B cells or hematopoietic stem cells. The MoFlo would greatly facilitate the identification and isolation of these types of cells by utilizing three lasers to simultaneously detect 8 colors as well as forward and side scatter. Additionally, the MoF1o can sort four populations at once using non-rectangular sort gates at speeds of 25,000 cells/second with substantially lower abort rates than a similar instrument from Becton-Dickinson. Thus, investigators will recover more precisely defined populations of cells in less time from fewer animals. Furthermore, investigations involving immunity to lung pathogens such as influenza, pneumocystis, tuberculosis, cryptococcus and pneumococcus will be significantly advanced by the availability of a HeNe laser, which allows the use of multiple red-shifted fluorochromes such as APC and Cy7-APC in the analysis and purification of cells in the lung or brain, which are highly autofluorescent in the conventional FITC and PE channels. The aerosol evacuation system on the MoFlo is also essential for these projects, since it will prevent the dispersal of infectious material from the sort stream area. Finally, the combination of enhanced optics and the Kr laser will allow Trudeau investigators to examine UV dependent parameters, such as low level Ca2+ flux, Hoescht staining of DNA, and some types of green fluorescent proteins. Therefore, we feel that the MoFlo is the instrument of choice for the Institute's investigators.